Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol’s impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity. [Display omitted] •Specific cholesterol-A2AAR interactions activate receptor in POPC lipid bilayers•Influence of sterols on agonist-bound A2AAR observed with cholesterol analogs•Cholesterol and anionic lipids share complementary roles in A2AAR activation•Different cholesterol interaction sites distinctly impact structure and function Ray and Thakur et al. use 19F-NMR to observe how cholesterol affects the conformational equilibria of the A2A adenosine receptor. The impact of cholesterol varies depending on the type of phospholipids present and differs at different receptor locations, affecting either structural stability or receptor signaling activity.